Aging is the most important risk-factor for late-onset Alzheimer's disease (AD), which represents approximately 95% of all cases of AD dementia. The second messenger ceramide regulates many of the biochemical events that occur during aging. Our Preliminary Data suggest that ceramide regulates the biogenesis of amyloid beta-peptide (Abeta), the first pathogenic event in the generation of senile (or amyloid) plaques. Such effect occurs in senescent-like conditions, is produced by a selective increase in beta cleavage of the amyloid precursor protein (APP), and is mediated by acetylation and molecular stabilization of the beta-secretase BACE1. Our results also show that the p75 neurotrophin receptor regulates the generation of both ceramide and Abeta in an age-dependent fashion. Finally, they show that ceramide levels, BACE1 steady-state levels, and Abeta generation increase progressively during aging. Such event is produced by an age-dependent activation of neutral sphingomyelinase (nSMase), and is inhibited by nSMase inhibitors. The long-term objectives of this application are to identify the up-stream and down-stream molecules that mediate the ceramide-dependent regulation of Abeta generation, and to evaluate the possibility that inhibitors of nSMase may serve as a novel therapeutic approach for the treatment of AD. Specific Aim 1 describes experiments aimed at the identification of the up-stream and down-stream molecules that mediate the ceramide-dependent regulation of Abeta generation. They will be performed using biochemical, cellular, and genetic approaches. The biochemical approach includes pharmacological inhibitors of the ceramide signaling pathways, whereas the genetic approach includes neuronal mutant cell lines, antisense oligonucleotides, and siRNA. Specific Aim 2 describes experiments aimed at the characterization of the mechanisms by which ceramide regulates the acetylation and stabilization of BACE1. They will analyze how ceramide affects the acetylation and turn-over of BACE1, and will include biochemical and genetic approaches, such as in vivo and in vitro acetylation, and site-directed mutagenesis. Specific Aim 3 will analyze whether ceramide increases the biogenesis of Abeta during aging, and whether nSMase inhibitors represent a novel therapeutic strategy for the treatment of AD. nSMase activity and ceramide levels will be analyzed in both aging neurons and organotypic brain cultures. The effects of nSMase inhibitors in AD pathology will be analyzed using "AD transgenic mice".